Diversity and Evolution of tularensis Gunnell et al.

The Genetic Diversity and Evolution of with Comments on Detection by PCR

Mark K. Gunnell1,3*, Byron J. Adams2 and Richard A. (McCoy and Chapin, 1912). Following the Bacterium Robison1 genus, it was subsequently placed in Pasteurella and later (Salomonsson, 2008). Finally in 1959, it was 1Department of and Molecular Biology, placed in a new genus, Francisella, in honor of Edward Brigham Young University, Provo, UT 84602, USA Francis, in which genus it resides today (Olsufjev et al., 2Department of Biology, and Evolutionary Ecology Laboratories, 1959). There are currently four recognized subspecies of Brigham Young University, Provo, UT 84602, USA Francisella tularensis: tularensis, holarctica, mediasiatica, 3Microbiology Branch, Life Sciences Division, Dugway and novicida. While the inclusion of novicida as a Proving Ground, Dugway, UT 84022, USA subspecies of F. tularensis is still contested (Larsson et al., *Corresponding Author: [email protected] 2009; Kingry and Petersen, 2014), much of the recent scientific literature, including Bergey’s Manual of http://dx.doi.org/10.21775/cimb.018.079 Systematic , recognizes this classification (Garrity, 2005). Abstract Francisella tularensis has been the focus of much research In 1950, the first novicida subspecies was isolated and over the last two decades mainly because of its potential characterized (Larson et al., 1955). This new isolate use as an agent of . F. tularensis is the resembled F. tularensis morphologically, but differed in that causative agent of zoonotic and has a worldwide it could ferment glucose, was not as virulent in humans, distribution. The different subspecies of F. tularensis vary in and did not cross-react with serum from rabbits inoculated their biogeography and , making early detection with killed F. tularensis. Based on these differences, the and diagnosis important in both the biodefense and public authors proposed the name (Larson et health sectors. Recent sequencing efforts reveal al., 1955). However, in the 1950s, researchers did not have aspects of genetic diversity, evolution and phylogeography the genetic tools which became available in later decades. previously unknown for this relatively small organism, and In the 1980s, DNA-DNA hybridization experiments between highlight a role for detection by various PCR assays. This F. tularensis and F. novicida demonstrated up to 92% review explores the advances made in understanding the homology (Hollis et al., 1989). Because of this high degree evolution and genetic diversity of F. tularensis and how of genetic similarity, it was proposed that F. novicida be these advances have led to better PCR assays for reclassified as a subspecies of F. tularensis . This detection and identification of the subspecies. reclassification was formally proposed in 2010 in the International Journal of Systematic and Evolutionary Introduction Microbiology (IJSEM) (Huber et al., 2010). This proposal Francisella tularensis is a small, non-motile, Gram-negative received a formal objection in IJSEM, contending that and is the causative agent of the zoonotic genetic similarity was not enough to reclassify F. novicida tularemia. This facultative intracellular as F. tularensis subsp. novicida, but that the phenotypic was first discovered in Tulare County California in 1911 differences were sufficient enough to justify separate where it caused a -like illness in local designation (Johansson et al., 2010). (McCoy and Chapin, 1912). F. tularensis is able to cause disease in rabbits, squirrels, and other , including Finally, in a rebuttal to the objection of Johansson et al., humans (Wherry and Lamb, 1914). The of F. Busse et al. (2010), stood by their initial recommendation for tularensis to humans is mediated through arthropod reclassification, asserting that the genetic similarity meets vectors such as and flies, by the ingestion of the definition of a subspecies (Wayne et al., 1987). contaminated food or water, or by inhalation of aerosolized Furthermore, Busse et al. acknowledge the phenotypic (Akimana and Abu Kwaik, 2011). F. tularensis differences between F. tularensis and F. novicida , but subsp. tularensis is highly infectious. It is estimated that an contend that the 11 phenotypic differences noted are not aerosol inoculation of as few as 10 organisms is sufficient sufficient enough for a new species (Busse et al., 2010). to cause disease in humans (McCrumb, 1961). Because of There are many other examples of bacteria with a greater its highly infectious nature, F. tularensis is considered a percentage of phenotypic differences which are classified as potential agent of bioterrorism and is categorized by the the same species (e.g. the various biovars of Centers for Disease Control and Prevention (CDC) as a fluorescens) (Busse et al., 2010). Despite this evidence, a Tier 1 (Dennis et al., 2001). formal reclassification has yet to occur. Based on the high genetic similarity, and taking into account the relatively few Through the years, the of Francisella has gone phenotypic differences, we also propose the reclassification through many changes. Upon its discovery, McCoy and of F. novicida as a subspecies of F. tularensis , and will refer Chapin named their new discovery Bacterium tularense to it as such throughout this work.

Curr. Issues Mol. Biol. (2016) 18: 79-92. horizonpress.com/cimb !79 Diversity and Evolution of Francisella tularensis Gunnell et al.

Each subspecies is predominantly associated with a include Type A.I and Type A.II, with the former generally specific geographic distribution and severity of disease. isolated from the eastern United States and the latter The subspecies tularensis is typically found in North generally isolated from the western United States America (Staples et al., 2006) while the subspecies (Johansson et al., 2004). This biogeographic separation is holarctica is found across much of the Northern correlated with the geographic distribution of specific Hemisphere (Johansson et al., 2004). The subspecies vectors, hosts, and other abiotic factors such as elevation mediasiatica has only been isolated from the central Asian and rainfall (Farlow et al., 2005; Oyston, 2008). The major republics of the former Soviet Union (Broekhuijsen et al., divisions of Type B tularensis also display geographic 2003) and the subspecies novicida has been isolated from structure, with Type B.I isolated from Eurasia, Type B.II and Australia (Hollis et al., 1989; Whipp et isolated from North America and Scandinavia, Type B.III al., 2003). Phylogenetic relationships among these isolated from Eurasia and North America, Type B.IV subspecies are inferred in Figure 1. isolated from North America and Sweden, and Type B.V isolated from Japan (Johansson et al., 2004). Unlike type A The two subspecies most associated with human disease tularensis, the distribution of Type B tularensis has not are tularensis and holarctica. These are often abbreviated been shown to correlate with the distribution of any specific simply as Type A and Type B tularensis, respectively. Type vectors (Farlow et al., 2005). A tularensis causes a more severe form of tularemia while the presentation of type B tularemia is somewhat milder The F. tularensis subsp. holarctica isolated from Japan was (Owen et al., 1964; Weiss et al., 2007). The subspecies first differentiated from other F. tularensis subspecies mediasiatica is fully virulent in mice, yet is believed to be of based on its ability to ferment glucose (Olsufjev and relatively mild virulence in humans (Broekhuijsen et al., Meshcheryakova, 1983). These isolates were further 2003; Champion et al., 2009). Similar to the subspecies differentiated by demonstrating a reduced virulence from mediasiatica, the subspecies novicida is fully virulent in the subspecies tularensis, displaying a virulence similar to mice, yet rarely causes disease in humans (Hollis et al., that of the subspecies holarctica (Sandstrom et al., 1992). 1989). As genomic tools became more widely available, this division was confirmed by microarray analysis Genetic analyses by multiple-locus variable-number (Broekhuijsen et al., 2003), restriction fragment length tandem repeat analysis (MLVA) has identified further sub polymorphism (RFLP) analysis (Thomas et al., 2003), and classifications and geographic structure of Type A and Type multiple-locus variable number tandem repeat analysis B tularensis. The major subdivisions of Type A tularensis (MLVA) (Johansson et al., 2004; Fujita et al., 2008).

Figure 1. Maximum likelihood tree inferring the phylogenetic relationships of the F. tularensis subspecies. Tree was constructed by concatenating 10 housekeeping genes (recA, gyrB, groEL, dnaK, rpoA1, rpoB, rpoD, rpoH, fopA, and sdhA) followed by alignment with Clustal W and generation of the tree with MEGA 5.2. Bootstrap values are indicated at the nodes except where support was less than 0.65. Figure 1. Maximum likelihood tree inferring the phylogenetic relationships of the F. tularensis subspecies. Tree was constructed by concatenating 10 housekeeping genes (recA, gyrB, groEL, dnaK, rpoA1, rpoB, rpoD, rpoH, fopA, and sdhA) followed by alignment with Clustal W and generation of Curr. Issues Mol. Biol. (2016) 18: 79-92. horizonpress.com/cimb !80 the tree with MEGA 5.2. Bootstrap values are indicated at the nodes except where support was less than 0.65.

Diversity and Evolution of Francisella tularensis Gunnell et al.

Genetic analyses have hinted that these isolates from (Samrakandi et al., 2004). While microarray and other Japan underwent a unique evolutionary process in a studies revealed valuable information about the regional restricted area, separate from other F. tularensis distribution and differences in virulence, complete genome subspecies (Fujita et al., 2008). Because of the phenotypic sequences reveal a more complete picture (Broekhuijsen differences, the genetic differences, and the apparent et al., 2003; Johansson et al., 2004; Samrakandi et al., isolated evolution, it has been proposed that these strains 2004). from Japan be classified as another subspecies of F. tularensis called F. tularensis subsp. japonica The first completed genome sequence of F. tularensis (Vivekananda and Kiel, 2006). However, since relatively yielded insights to previously undiscovered features of its few isolates from Japan have been analyzed, we genetic makeup. Some of the genetic features discovered recommend that this designation not be adopted at this included previously uncharacterized virulence genes time. encoding type IV pili and iron acquisition systems (Larsson et al., 2005). The complete sequence also revealed a Genetic Diversity duplication of an approximately 30 kb region previously The first complete genome of Francisella tularensis was identified as a containing 17 open sequenced in 2005 (Larsson et al., 2005). This first reading frames (ORFs), perhaps shedding light on the sequence was the classical type strain of Francisella enhanced virulence of Type A tularensis (Nano et al., 2004; tularensis subsp. tularensis representing the Type A.I sub Larsson et al., 2005; Nano and Schmerk, 2007). Finally, classification. Since then, numerous other whole and analysis of this genome indicated the loss of several partial of F. tularensis have been sequenced: F. biosynthetic pathways, which helps explain the fastidious tularensis subsp. holarctica strain OSU18 (Type B) nutritional requirements of F. tularensis and suggests the (Petrosino et al., 2006), a European isolate of Type A need to infect a host during its life cycle (Larsson et al., tularensis (Chaudhuri et al., 2007), F. tularensis subsp. 2005). novicida strain U112 (Rohmer et al., 2007), a Type A.II tularensis (WY96-3418) (Beckstrom-Sternberg et al., The first comparative genomic study of F. tularensis was of 2007), F. tularensis subsp. mediasiatica (Larsson et al., the Type A (Schu S4) and Type B (OSU18) strains. This 2009) and at least 10 more comprising the 4 subspecies of study revealed an extensive genomic similarity of 97.63%, F. tularensis (Barabote et al., 2009; Nalbantoglu et al., indicating that the differences in virulence between the two 2010; Modise et al., 2012; Svensson et al., 2012). With the strains are likely not due to large differences in gene advent of improved massively parallel sequencing content (Petrosino et al., 2006). This degree of sequence technologies, more genomes continue to be sequenced at identity was confirmed among the remaining subspecies as an ever-increasing rate (La Scola et al., 2008). In all, there well (Rohmer et al., 2007; Champion et al., 2009; Larsson are currently 16 complete genomes of Francisella et al., 2009). Perhaps the most striking difference between tularensis deposited in GenBank and even more partial these two strains is the vast amount of genomic genomes. This collection of genomic information allows for rearrangement. These rearrangements can mostly be the comparative analysis of these genomes and provides attributed to homologous recombination using insertion (IS) insight into the evolution of F. tularensis genome elements (Petrosino et al., 2006). architecture. After the genome sequence of F. tularensis subsp. novicida Even before the first Francisella genome was completed in was complete, a 3-way comparison between three of the 2005, studies analyzing the genomic diversity of F. subspecies (tularensis, holarctica, and novicida) was tularensis were plentiful. Because of its potential use as a possible. Again, a high degree of sequence identity among bioweapon and for public health reasons, rapid the subspecies was confirmed, as was the large amount of identification of F. tularensis became paramount (Dennis et genomic rearrangement (Rohmer et al., 2007). Even al., 2001). Early DNA based techniques focused on 16S though the length and the gene content of the novicida rDNA typing. This proved difficult since among the 4 subspecies (1.91 Mb and 1,731 protein coding genes) are subspecies, the 16S rDNA genes exhibit between 98.5 - both greater than that of the tularensis subspecies (1.89 99.9% similarity, the result of only 6 nucleotide differences Mb and 1,445 protein coding genes) and the holarctica among the most divergent strains (Forsman et al., 1994). subspecies (1.89 Mb and 1,380 protein coding genes), Other DNA based techniques for identification such as these human pathogenic strains contain 41 genes which PCR, which is both rapid and accurate, helped spur further the non-human pathogenic strains (novicida) do not interest in the genetic diversity of the F. tularensis (Rohmer et al., 2007). Initial comparisons of these subspecies (Broekhuijsen et al., 2003; Pohanka et al., genomes revealed that the human pathogenic strains carry 2008). A genome wide microarray that analyzed 27 strains 2 copies of the Francisella Pathogenicity Island (FPI) while of all four subspecies confirmed the limited genetic the non human pathogenic strains carry only 1 copy, variation within the subspecies, but identified 8 variable shedding further light on the differences in virulence among regions that were used to develop a subspecies-specific the subspecies (Nano and Schmerk, 2007). PCR assay (Broekhuijsen et al., 2003). Another microarray study analyzing the genetic diversity of 11 Type A isolates Many studies have been completed comparing the various and 6 Type B isolates from various localities around the subsets of available F. tularensis genomes. A comparison United States identified 13 regions of difference, including of the genomes of two holarcitca subspecies, the live segments of several genes with implications for virulence strain (LVS) and strain FSC200, sought to uncover

Curr. Issues Mol. Biol. (2016) 18: 79-92. horizonpress.com/cimb !81 Diversity and Evolution of Francisella tularensis Gunnell et al. the mode of attenuation for LVS (Rohmer et al., 2006), et al., 2009; Nalbantoglu et al., 2010). Another genome which was attenuated through the repeated passage of a comparison of a Type A.I clinical isolate to the Schu S4 holarctica strain between the 1930s and 1950s in the genome showed that except for some minor changes, the former Soviet Union (Green et al., 2005; Petrosino et al., genomes were virtually identical, suggesting a high degree 2006). The genomes of the LVS and FSC200 strains differ of sequence conservation within the Type A.I subgroup by only 0.08% but the LVS strain was able to confer (Nalbantoglu et al., 2010). The genome of another Type A.I immunity to with F. tularensis subsp. tularensis in strain (TI0902) isolated from a in Virginia, United BALB/c mice (Green et al., 2005; Rohmer et al., 2006). States, is also highly similar to Schu S4 as it only differs by While the exact nature of genomic modifications leading to 103 SNPs (Modise et al., 2012). Other researchers LVS attenuation were not found, comparison with other compared a European isolate of Type A.I tularensis (which more virulent Type A strains revealed some candidate is typically restricted to North America) to Schu S4 and genes which could be targeted in the development of a found that the two were virtually identical, with only 8 SNP future vaccine (Rohmer et al., 2006). When the sequence and 3 variable number tandem repeat (VNTR) differences of F. tularensis subsp. holarctica FTNF002-00 was (Chaudhuri et al., 2007). The fact that these two strains are completed and compared to both LVS and the OSU18 so alike suggests that the European isolates are strains, it was found to have greater than 99.9% sequence descended from the Schu S4 strain and did not evolve similarity (Barabote et al., 2009). Other studies have shown independently in (Chaudhuri et al., 2007). a stable genome architecture among Type B strains, but FTNF002-00 carries a 3.9 kb inversion compared to other The completion of a fourth subspecies genome of F. Type B strains (Petrosino et al., 2006; Dempsey et al., tularensis, the mediasiatica subspecies, enabled full 2007; Barabote et al., 2009). genome comparisons of the four subspecies of F. tularensis. It was demonstrated that the subspecies Other whole genome comparisons focused on comparing mediasiatica and tularensis are highly similar, which raises different strains of Type A tularensis. A comparison more questions about their differences in virulence between F. tularensis subsp. tularensis Schu S4 (Type A.I) (Olsufjev and Meshcheryakova, 1982; Broekhuijsen et al., and WY96-3481 (Type A.II) revealed only one whole gene 2003; Larsson et al., 2009). Phylogenetic analysis of the difference, a hypothetical protein with an unknown function complete genomes of the subspecies mediasiatica also (Beckstrom-Sternberg et al., 2007). Despite the fact that demonstrated that it is a monophyletic taxon of F. these two strains are very closely related, there were still tularensis, contradicting previous evidence suggesting that many other differences, including numerous single the subspecies mediasiatica was not a member of the F. nucleotide polymorphisms (SNPs), small indels, differences tularensis clade (Nübel et al., 2006). However, since in IS elements, and even 31 large chromosomal isolates of the mediasiatica subspecies are rare, it is rearrangements (Beckstrom-Sternberg et al., 2007). Many difficult to know the true genetic diversity within the of the chromosomal rearrangements are frequently subspecies. Figure 2 shows the overall genome bordered by IS elements, providing a mechanism for the architecture of representative strains of F. tularensis , translocations (Beckstrom-Sternberg et al., 2007; Larsson

A

B

C

D

Figure 2. Whole Figuregenome alignment 2. Whole of representative genome strains alignment from each of of the representative four subspecies of Francisella strains tularensis from using each Mauve of (Darlingthe four et al., 2004) highlighting differences in the macro genome architecture relative to the reference strain (A). Colored blocks represent homologous sections of each genome. A) F. tularensis subsp. tularensis Schu S4.subspecies B) F. tularensis subsp.of Francisella holarctica LVS. tularensisC) F. tularensis subsp.using mediasiatica Mauve FSC147. (Darling D) F. tularensiset al., 2004subsp. novicida) highlighting U112. differences in the macro genome architecture relative to the reference strain (A). Colored blocks represent homologous sections of each genome. A) F. tularensis subsp. tularensis Curr. Issues Mol. Biol. (2016) 18: 79-92. horizonpress.com/cimb !82 Schu S4. B) F. tularensis subsp. holarctica LVS. C) F. tularensis subsp. mediasiatica FSC147. D) F. tularensis subsp. novicida U112.

Diversity and Evolution of Francisella tularensis Gunnell et al. highlighting the large-scale genomic rearrangements screening tool and further testing is required to differentiate between the subspecies. between the organisms comprising the assay. Furthermore, since the genome of Variola Major (the causative agent of The evolution of the Francisellacaea is complicated by the Smallpox) is so highly regulated, testing was completed discovery of Francisella-like endosymbionts (FLEs) of ticks, with a plasmid control containing a small segment of the which have an unknown pathogenicity in humans Variola Major genome (He et al., 2009). (Niebylski et al., 1997; Scoles, 2004; Goethert and Telford, 2005; Dergousoff and Chilton, 2012). While these Real-time PCR is known for being efficient and sensitive, endosymbionts lack sufficient evidence to be classified as but is not ideal for multiplexing beyond a 4- or 6-plex F. tularensis, they are similar enough to cross react with reaction because of the limited number of fluorescent many molecular-based methods of detection (Szigeti et al., channels available on most instrument platforms (Varma- 2014). Because of the potential to misidentify FLEs as F. Basil et al., 2004; Skottman et al., 2007). Researchers tularensis, which could impact the diagnosis of tularemia in have overcome this limitation by using modified primers to public heath settings, many have cautioned about the use bind the PCR products of a 15-plex reaction to fluorescent of PCR assays for the detection of F. tularensis (Kugeler et beads that can then be analyzed by a flow cytometer for al., 2005; Sreter-Lancz et al., 2009). Despite this caution, the simultaneous detection of 11 with similar PCR remains the standard of practice for the detection and sensitivities to real-time reactions (Hsu et al., 2013). While identification of F. tularensis subspecies (Pohanka et al., effective, flow cytometers can be large, difficult to use, and 2008). costly. The Luminex Corporation (Austin, TX) has developed a similar, yet easier to use technology in their Detection MAGPIX® system. Rather than a flow cell, the MAGPIX® The ability to accurately detect and diagnose F. tularensis uses a magnet to capture fluorescently labeled magnetic infection carries significant implications in public health and beads and a CCD camera to capture images of up to 50 bioterror (Dennis et al., 2001; Gunnell et al., 2012). different analytes (Bergval et al., 2012; Munro et al., 2013). Because of the different pathogenic profiles and Because of its relatively low cost and ease of use, the biogeography of the various subspecies of F. tularensis, it MAGPIX® may be more ideally suited for integration in is important to be able to accurately discriminate among clinical labs for the simultaneous detection of multiple them (Gunnell et al., 2012). Polymerase chain reaction pathogens (Bergval et al., 2012). (PCR) has become the method of choice for the identification of various pathogens because it is rapid, While it may be useful to detect broad categories of sensitive and highly specific (Foroshani et al., 2013; Sting pathogens, because of the virulence status of various et al., 2013; Celebi et al., 2014). Detection and subspecies of F. tularensis, it is also important to be able to differentiation of the subspecies of F. tularensis by PCR is differentiate among them as well. Using the tul4 gene and complicated by the lack of significant variability in their variations in the pilA gene, researchers were able to genomes (Forsman et al., 1994; Petrosino et al., 2006). differentiate the four subspecies of F. tularensis Various methods for the detection of F. tularensis have (Kormilitsyna et al., 2013). Another study used suppression been reviewed in the last decade, however much more subtractive hybridization (SSH) to identify regions of work has since been completed on the detection of F. difference between the genomes of Type A.I and Type A.II tularensis using PCR (Splettstoesser et al., 2005; Pohanka tularensis. This information was used to create a et al., 2008). conventional PCR assay to differentiate between Type A.I, Type A.II, Type B, and F. tularensis subsp. novicida isolates Conventional PCR (Molins-Schneekloth et al., 2008). Later, this same assay Since 2008, research on the use of conventional PCR for was adapted to a real-time PCR platform (Molins et al., the detection of F. tularensis has dropped off considerably, 2009). with only a handful of publications on the subject. In alignment with an earlier review (Splettstoesser et al., Real-time PCR 2005), the gene tul4 was a popular choice to detect all Real-time PCR is a popular choice for the detection of F. subspecies of F. tularensis (He et al., 2009; Kormilitsyna et tularensis because it is sensitive, reliable, cost-effective, al., 2013). Since F. tularensis is a potential agent of and eliminates the need for time consuming gels, though bioterrorism, some assays included the multiplex detection this time commitment has been significantly reduced with of other biothreat agents. One such study developed two the introduction of rapid dry gels (Zasada et al., 2013). A multiplex assays to detect “Tier 1” select agents; one assay popular method of real-time PCR incorporates the use of for DNA based organisms (Variola Major, SYBR Green which will fluoresce upon binding double anthracis, pestis, Francisella tularensis, and stranded DNA. Thus, the fluorescent signal will increase as Varicella zoster virus) and another assay with a reverse PCR progresses and more amplicons are synthesized. transcriptase for RNA based viruses ( virus, Lassa SYBR green is a popular alternative to other real-time virus, Rift Valley fever, Hantavirus Sin Nombre and technologies because of its relatively low cost (Sellek et al., the four serotypes of Dengue virus) (He et al., 2009). A 2008). However, it is not ideal for multiplex reactions since major drawback to these multiplex assays however, is the the dye will bind to all double stranded DNA in the reaction use of a reporter dye and a colormetric detection system, and produce a fluorescent signal. Sellek et al. (2008) because a positive result is unable to distinguish between developed an assay to detect F. tularensis from soil using the agents. The assay is intended only as a broad the tul4 gene, previously used in conventional PCR assays

Curr. Issues Mol. Biol. (2016) 18: 79-92. horizonpress.com/cimb !83 Diversity and Evolution of Francisella tularensis Gunnell et al.

(He et al., 2009; Kormilitsyna et al., 2013). However, the (Yang et al., 2008) or other organisms with similar disease assay was only validated with F. tularensis subsp. presentations (Angelakis et al., 2009), while others were holarctica and subsp. novicida. Lacking were used solely for the differentiation of subspecies and representatives from the subsp. tularensis and subpopulations of F. tularensis (Molins et al., 2009; Birdsell mediasiatica. Furthermore, positive fluorescent signals et al., 2014b). The advantage of using a single-copy gene were obtained from other non-related bacteria. These were for detection is the ability to quantify the amount of the later ruled out as true positives after analyzing the PCR agent, which can be useful in clinical and diagnostic products on a gel and finding only primer dimers (Sellek et settings (Abril et al., 2008). Conversely, multicopy-genes al., 2008). such as the 16S rRNA gene and the ISFtu2 gene should achieve lower detection limits, which is ideal given the low Genome comparisons aided the development of SYBR infectious dose of F. tularensis (McCrumb, 1961; Yang et green assays (Pandya et al., 2009; Svensson et al., 2009; al., 2008; Simsek et al., 2012). A significant drawback of Woubit et al., 2012). Woubit et al. (2012) compared several using the 16S rRNA gene for detection is that since it is so genomes from the , Francisella, , conserved, there is some cross reactivity with near , , and Yersinia genera to develop a series of neighbors and other Francisella-like species, requiring 27 assays to detect and differentiate these common food further confirmatory analyses (Forsman et al., 1994; Yang and biothreat pathogens. With respect to Francisella, the et al., 2008). assays were so specific that assays intended to detect all subspecies of Francisella were only able to detect the Multiplex real-time TaqMan® assays incorporate the added tularensis and novicida subspecies (Woubit et al., 2012). convenience of running multiple reactions in a single tube using probes labeled with various fluorophores. However, The propensity of PCR assays to cross-react with as mentioned previously, multiplexing with TaqMan® environmental, non-pathogenic Francisella or other closely assays is generally limited to a 4- or 6 plex reaction related organisms (Kugeler et al., 2005) requires the because of the limited number of fluorescent channels on development of more specific assays to avoid false the instruments (Varma-Basil et al., 2004; Skottman et al., positives or incorrect diagnoses. To solve this problem, 2007). One multiplex assay is a 2-plex assay designed results from resequencing microarrays were compared to from genome comparisons to detect the four subspecies of identify SNPs along the phylogeny of F. tularensis and build F. tularensis but does not differentiate among them. real-time PCR assays capable of differentiating Type A.I, Another multiplex assay is capable of differentiating the A.II, A.Ia, A.Ib, Type B.I, and B.II tularensis (Pandya et al., four F. tularensis subspecies with only a 3-plex assay. This 2009). Similarly, another group analyzed publically assay was developed using both unique and shared available whole genome sequences to identify defining genome regions among the subspecies with the addition of SNPs and small insertion/deletion elements (INDELs) to a scoring matrix (Gunnell et al., 2012). design a series of 35 assays capable of distinguishing the four subspecies of F. tularensis and the major subtypes of Since F. tularensis has the potential to be used as a Type A and Type B tularensis, including Type A.I, A.II, and bioweapon, a commercial market has arisen for field-ready B.I, B.II, B.III, B.IV, and B.V (Svensson et al., 2009). Both detection of biothreat agents, including , assays were able to accurately assign isolates to the Francisella tularensis, Yersinia pestsis, Brucella species, correct subspecies and clade while avoiding any cross- and others. A comparison of one such commercial reactivity to near neighbors (although the former includes instrument, the RAZOR®, (BioFire Defense; previously only one novicida strain in the analysis). Idaho Technologies, Salt Lake City, UT) and another instrument designed for laboratory use, the Applied Another method for the real-time detection of F. tularensis Biosystems 7300/7500 system (Thermo Fisher Scientific, is the 5’ nuclease or TaqMan® assay. These assays Grand Island, NY) used assays developed for B. anthracis, incorporate fluorescently labeled DNA probes specific to Brucella species, F. tularensis, and Y. pestis, comparing the template DNA resulting in even more specific sensitivities and specificities of the two platforms. Results identification than the SYBR Green assays, eliminating the showed that for all agents, the sensitivities were between need to perform a melt curve analysis. Strategies for 10-100 fg of target DNA per reaction, and no cross single-plex real-time assays for the detection of F. reactivity was observed with other closely related bacteria tularensis with TaqMan® assays are varied. Gene targets (Matero et al., 2011). Run time on the RAZOR® was include a gene for an outer membrane protein, FopA, a notably shorter than that of the 7300/7500 instrument. single-copy gene for detection and quantification of all subspecies of F. tularensis (Abril et al., 2008), the 16S Another diagnostic tool, the FilmArray® system (BioFire rRNA gene to detect all subspecies of F. tularensis (Yang et Defense, Salt Lake City, UT), uses a lab-in-a-pouch al., 2008; Angelakis et al., 2009), the insertion element approach to process raw samples and detect 17 biothreat ISFtu2, which is unique to Francisella species (Simsek et pathogens with an array of single-plex real-time PCR al., 2012), intergenic regions of differentiation to distinguish assays in about an hour (Seiner et al., 2013). An evaluation Type A.I from Type A.II tularensis (Molins et al., 2009), and of the Biothreat Panel using DNA samples from B. SNP-based assays to differentiate the species and anthracis, F. tularensis, and Y. pestis indicated sensitivities subspecies of Francisella isolates (Birdsell et al., 2014b). of 250 genome equivalents or lower and the authors Some assays can be used in concert with others to detect conclude that the system is both sensitive and selective a wide variety of agents. These include biothreat agents (Seiner et al., 2013). However, since the FilmArray®

Curr. Issues Mol. Biol. (2016) 18: 79-92. horizonpress.com/cimb !84 Diversity and Evolution of Francisella tularensis Gunnell et al. system is designed to be a complete sample to answer strain typing of B. anthracis, F. tularensis, and Y. pestis by system, sensitivities may vary when tested with whole interrogating 10 loci per pathogen (Turingan et al., 2013). organisms in different matrices like blood or serum rather While sequencing assays provide some promise for the than purified DNA. rapid detection and classification of F. tularensis, there is a noticeable lack of information on the sensitivity or detection Another evaluation compared the FilmArray® system with limits of these assays. In the world of clinical diagnostics TaqMan® Array Cards developed for the detection of and biodefense, the ability to detect low quantities of F. biothreat agents (Rachwal et al., 2012; Weller et al., 2012). tularensis and other agents is paramount. Here, researchers tested for B. anthracis, F. tularensis, and Y. pestis in the blood of murine infection models. Results Evolution showed that was the most sensitive means of Numerous studies have been conducted on the evolution detection followed by the FilmArray and Array Cards for B. of the subspecies of F. tularensis to define specific clades anthracis, and F. tularensis. All three methods and to reveal their evolutionary history. Before next demonstrated similar detection levels for Y. pestis (Weller generation whole genome sequencing was widely et al., 2012). While blood culture was the most sensitive available, various techniques were used to recover the means of detection for two of the three agents tested, it phylogenetic relationships among strains of F. tularensis, requires much more time for detection compared to the such as microarrays (Broekhuijsen et al., 2003; PCR assays. Each of these methods for detection carries Samrakandi et al., 2004), MLVA (Johansson et al., 2004), drawbacks and benefits and must be weighed and sequencing specific genes or other genetic loci appropriately to ensure the best possible outcome. (Svensson et al., 2005; Nübel et al., 2006). One of the earliest of these studies produced a phylogenetic tree in Other PCR assays which the subspecies tularensis and mediasiatica shared a Recently, other PCR-based assays have been developed major clade along with the Japanese isolates of the for the detection of F. tularensis and other bacteria. One holarctica subspecies (Broekhuijsen et al., 2003). A later such assay involves analyzing PCR products with analysis provided better resolution, differentiating the electrospray ionization-mass spectrometry (ESI-MS). In tularensis and mediasiatica subspecies, and grouping the this technique, the actual base composition of the PCR Japanese isolates of the holarctica subspecies with the products are identified and compared to a library of other holarctia subspecies (Johansson et al., 2004). These sequences for identification rather than relying on the authors also determined that F. tularensis subsp. holarctia fluorescent signal obtained from real-time PCR (Jacob et appears to have recently spread globally from a single al., 2012). This PCR/ESI-MS technique has been applied to geographic origin, while F. tularensis subsp tularensis the wide-spread identification of biothreat agents, appears to have experienced most of its evolutionary respiratory pathogens, and other and history in North America, and may even have originated in viruses (Jacob et al., 2012; Jeng et al., 2013). Others have the central United States (Birdsell et al., 2014a). However, used this technology specifically for identifying F. tularensis F. tularensis subsp. tularensis is now clearly distributed from natural sources (Whitehouse et al., 2012) and even beyond North America into parts of Europe (Chaudhuri et for typing the subspecies of F. tularensis (Duncan et al., al., 2007). 2013). The finding that the subspecies holarctica recently spread Recombinase Polymerase Amplification (RPA) is a PCR- from a single origin seems likely because of the small like assay in which amplification is carried out at one amount of genetic diversity within the subspecies, that has temperature (isothermal) instead of cycling temperatures been identified by a variety of molecular methods (Farlow as in PCR. Recently, RPA assays have been applied to the et al., 2005; Dempsey et al., 2006; Rohmer et al., 2006; detection of F. tularensis and other biothreat agents (Euler Keim et al., 2007; Larsson et al., 2007). However, the et al., 2012; Euler et al., 2013; del Rio et al., 2014). Two of precise area of origin of the subspecies holarctica is these assays showed comparable sensitivities to real-time unknown. Based on phylogenetic analyses, there are two PCR assays with an instrument run time of about 10 competing hypothesis as to its origin: 1) the subspecies minutes (Euler et al., 2012; Euler et al., 2013). A third holarctica originated in Asia or 2) the subspecies holarctica assay using electrochemical detection rather than originated in North America before spreading around the fluorescent probes seemed less sensitive than other Northern Hemisphere (Vogler et al., 2009). There appears assays, with detection levels on the order of 104 copies/µL to be more evidence for the origination of the subspecies (del Rio et al., 2014). holarctica in North America, though this may be due to the lack of Asian isolates for analysis. Regardless, it appears Finally, as the cost of sequencing continues to fall, more that the holarctica subspecies is a highly fit clone that sequencing-based detection assays are being used to originated from a single source and spread throughout the detect biological agents such as F. tularensis. One such Northern Hemisphere (Keim et al., 2007; Vogler et al., assay used a pyrosequencing method to sequence the 2009). However, if F. tularensis subsp. tularensis originated variable region of 16S rDNA to identify and group F. in North America (Johansson et al., 2004; Birdsell et al., tularensis isolates by subspecies (Jacob et al., 2011). The 2014a) and the subspecies holarctica is descended from results from analyzing the SNPs in 16S rDNA are more the tularensis subspecies (Svensson et al., 2005), then it distinctive than SNP analysis from real-time PCR. Another seems likely that the subspecies holarctica may have sequencing assay was multiplexed for the detection and originated in North America as well. This hypothesis is

Curr. Issues Mol. Biol. (2016) 18: 79-92. horizonpress.com/cimb !85 Diversity and Evolution of Francisella tularensis Gunnell et al. supported by the fact that sequences of various Schmerk, 2007). However, because the mediasiatica housekeeping genes and some outer membrane proteins subspecies is so rare, assessments of its true genetic from the subspecies tularensis and holarctica align well, diversity must be considered preliminary. while those from the subspecies novicida and mediasiatica do not (Nübel et al., 2006). There are many pros and cons to the various PCR detection methods and the individual user’s needs should It is generally accepted that F. tularensis subsp. novicida is dictate which method to use. Conventional PCR is easy the oldest of the F. tularensis subspecies and evidence and inexpensive but is known for being time consuming suggests that F. tularensis subsp. novicida and Francisella because of the need to run gels. However, since the philomiragia share a common, aquatic ancestor (Svensson introduction of rapid dry gels, the time commitment usually et al., 2005; Sjödin et al., 2012; Zeytun et al., 2012). These associated with gels has been shortened considerably. two species are generally considered non-pathogenic to Utilizing fast PCR technology in combination with rapid dry humans. However, their association with aquatic sources is gels, it is possible to get a result in approximately 50 further substantiated in that documented human minutes (Zasada et al., 2013). In general, conventional by these two species have occurred in near-drowning PCR has fallen out of favor with many researchers. victims (Hollis et al., 1989; Wenger et al., 1989). However, this approach allows for large multiplex reactions Furthermore, F. philomiragia contains one copy of the FPI, for the detection of many organisms at once, especially similar to F. tularensis subsp. novicida while the remaining when coupled with another detection system such as the subspecies of F. tularensis contain 2 copies (Nano and MAGPIX® (Bergval et al., 2012; Munro et al., 2013). Schmerk, 2007; Zeytun et al., 2012). Real-time PCR is one of the most popular methods for Molecular evidence suggests that the four subspecies of F. detection because it is simple, cost effective, and sensitive. tularensis have evolved by vertical descent (Svensson et SYBR Green assays are inexpensive and accurate and al., 2005). A common method of acquiring genetic variation can even be multiplexed with the incorporation of a melting in bacteria is through . This is well curve analysis. TaqMan® assays are more expensive than documented in many species of bacteria, and especially in SYBR Green assays, but carry an additional layer of the conference of resistance (Bliven and Maurelli, specificity with the sequence of the probe. Multiplexing with 2012; Turner et al., 2014; Dunlop et al., 2015; Ying et al., TaqMan® assays is possible, but usually only up to a 4- or 2015). However, in the subspecies of F. tularensis, genetic 6-plex because of the limited number of available variation, including antibiotic resistance seems to have fluorescent channels on most instruments (Varma-Basil et arisen by rather than the acquisition of new genes al., 2004; Skottman et al., 2007). The limited amount of through horizontal gene transfer (Gestin et al., 2010; multiplexing with TaqMan® assays can be overcome by Siddaramappa et al., 2012; Sutera et al., 2014). setting up an array of single-plex reactions similar to the FilmArray® system (Seiner et al., 2013). An in silico analysis has recently shown that the non human-pathogenic F. tularensis subsp. novicida possesses Many current PCR assays lack the specificity to a CRISPER/Cas system to defend against invading genetic differentiate between environmental, non-pathogenic elements. This finding further supports the hypothesis that Francisella and other closely related organisms such as mutation is responsible for much of the evolution of F. FLEs (Kugeler et al., 2005; Szigeti et al., 2014). Perhaps in tularensis (Gallagher et al., 2008; Schunder et al., 2013). these situations, it would be wise to use whole genome Analyses of the other three virulent subspecies of F. sequencing assays for the detection of Francisella tularensis (tularensis, holarctica, and mediasiatica), reveal subspecies (Jacob et al., 2011; Turingan et al., 2013). that the genes responsible for the CRISPER/Cas system are non-functional (Schunder et al., 2013). This is As whole genome sequencing has become more widely somewhat puzzling since deletion of the CRISPER/Cas available, genome comparisons between the subspecies of system in other pathogens such as meningitidis, F. tularensis are possible and shed further light on the Camphylobacter jejuni, pneumophila, and genetic diversity and evolution of this pathogen. It is result in decreased virulence. It apparent that the more virulent subspecies of F. tularensis is hypothesized that in the case of F. tularensis, other have evolved from F. tularensis subsp. novicida primarily by in the genome have compensated for the genomic decay, genomic rearrangements, and the degeneration of the CRISPER/Cas system in the virulent duplication of the FPI (Rohmer et al., 2007). Many of the subspecies of F. tularensis (Sampson and Weiss, 2013). interrupted genes (pseudogenes) in the virulent subspecies of F. tularensis are metabolic genes, further supporting an Concluding Remarks intracellular life cycle, while other interrupted genes include The genetic diversity of the subspecies of F. tularensis secreted effector proteins that may have led to excessive appears to be quite limited. Genome comparisons among virulence, furthering the patho-adaption of F. tularensis as the subspecies reveal similarities greater than 95% an intracellular pathogen (Hager et al., 2006; Larsson et (Champion et al., 2009; Larsson et al., 2009). Many of the al., 2009; Siddaramappa et al., 2011; Bliven and Maurelli, differences in the genomes of F. tularensis are large-scale 2012). genomic rearrangements and a duplication of the pathogenicity island in the tularensis, holarctica, and mediasiatica subspecies (Petrosino et al., 2006; Nano and

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Acknowledgements to the subspecies rank of Francisella tularensis - We thank Dr. Angelo Madonna of Dugway Proving Ground response to Johansson et al. International Journal of for his guidance and leadership in the production of this Systematic and Evolutionary Microbiology 60, work. 1718-1720. Celebi, B., Kilic, S., Yesilyurt, M., and Acar, B. (2014). References Evaluation of a newly-developed ready-to-use Abril, C., Nimmervoll, H., Pilo, P., Brodard, I., Korczak, B., commercial PCR kit for the molecular diagnosis of Seiler, M., Miserez, R., and Frey, J. (2008). Rapid Francisella tularensis. Mikrobiyoloji Bulteni 48, 135-142. diagnosis and quantification of Francisella tularensis in Champion, M.D., Zeng, Q.D., Nix, E.B., Nano, F.E., Keim, organs of naturally infected common squirrel monkeys P., Kodira, C.D., Borowsky, M., Young, S., Koehrsen, M., (Saimiri sciureus). Veterinary Microbiology 127, 203-208. Engels, R., et al. (2009). Comparative genomic Akimana, C., and Abu Kwaik, Y. 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